XB130 Deficiency Enhances Lipopolysaccharide-Induced Septic Response and Acute Lung Injury

www.impactjournals.com/oncotarget/ Oncotarget, Vol. 7, No. 18

XB130 deficiency enhances lipopolysaccharide-induced septic response and acute lung injury

Hiroaki Toba1, Tereza Tomankova1, Yingchun Wang1, Xiaohui Bai1, Hae-Ra Cho1, Zhehong Guan1, Oyedele A. Adeyi2, Feng Tian1, Shaf Keshavjee1,3,4, Mingyao Liu1,3,4 1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, Universal Health Network, University of Toronto, Toronto, Ontario, Canada 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada 3Department of Surgery, University of Toronto, Toronto, Ontario, Canada 4Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Correspondence to: Mingyao Liu, e-mail: [email protected] Keywords: cell death, cell proliferation, inflammatory response, septic response, transgenic mouse Received: August 31, 2015 Accepted: March 08, 2016 Published: March 23, 2016

ABSTRACT XB130 is a novel oncoprotein that promotes cancer cell survival, proliferation and migration. Its physiological function in vivo is largely unknown. The objective of this study was to determine the role of XB130 in lipopolysaccharide (LPS)-induced septic responses and acute lung injury. LPS was intraperitoneally administrated to Xb130 knockout (KO) and wild type (WT) mice. There was a significant weight loss in KO mice at Day 2 and significantly higher disease scores during the 7 days of observation. The levels of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, interleukin-6 and interleukin-10 in the serum were significantly higher in KO mice at Day 2. In KO mice there were a significantly higher lung injury score, higher wet/dry lung weight ratio, more apoptotic cells and less proliferative cells in the lung. Macrophage infiltration was significantly elevated in the lung of KO mice. There was significantly increased number of p-GSK-3β positive cells in KO mice, which were mainly neutrophils and macrophages. XB130 is expressed in alveolar type I and type II cells in the lung. The expression in these cells was significantly reduced after LPS challenge. XB130 deficiency delayed the recovery from systemic septic responses, and the presence of XB130 in the alveolar epithelial cells may provide protective mechanisms by reducing cell death and promoting cell proliferation, and reducing pulmonary permeability.

INTRODUCTION RET/PTC oncogenic kinase in thyroid cancer cells [6]. XB130 protein expression was not associated with the XB130 is a novel adaptor protein for signal postoperative prognosis of patients with hepatocellular transduction [1, 2]. Due to its high molecular similarity carcinoma [11]. By contrary, XB130 expression was with actin filament associated protein (AFAP), XB130 is an independent prognostic factor in human esophageal also known as actin filament associated protein 1-like 2 squamous cell carcinoma [12]. In gastric cancer, reduced (AFAP1L2) [3, 4]. XB130 regulates cell proliferation and XB130 protein expression is a prognostic biomarker for cell cycle progression, prevents cell death, and promotes shorter survival and a higher recurrence rate [13]. On the cell migration, through its interaction with Src protein other hand, in human ductal breast cancer, XB130 was tyrosine kinase, and its binding with p85α, the regulatory present in the cytoplasm of malignant cells, but not in the subunit of PI3K, subsequently activating the PI3K/Akt normal breast cells, and positive XB130 expression was an pathway [1, 3–9]. independent risk factor for overall survival and recurrence XB130 was found in colorectal cancer cells as a Src free survival [14]. XB130, thus, may play different role in family kinase target [10], and then found as a target of tumorigenesis depending upon the types of cancers.

www.impactjournals.com/oncotarget 25420 Oncotarget Since most of cell biology studies on XB130 were disease scores, a marker of the severity of systemic sepsis, conducted in cancer cells, the physiological function of were significantly higher in Xb130 KO mice during the XB130 is largely unknown. Recently, there is a report 7 days of observation period (Figure 1C). that translocation of XB130 from cytoplasm to micro- LPS stimulation induced rapid significant increase filamentous structure is involved in NNK (a cigarette in serum levels of monocyte chemoattractant protein-1 smoking component) induced migration of normal (MCP-1), interleukin-6 (IL-6), tumor necrosis factor- human bronchial epithelial cells [8]. Moreover, the alpha (TNF-α), and IL-10 in both Xb130 KO and WT interaction between XB130 and another adaptor protein, mice (Figure 2A and 2B). It also rapidly and significantly Tks5, mediates EGF and serum-induced proliferation increased serum levels of IL-1ß, IL-12p70, IL-13, IL-17 and survival of human bronchial epithelial cells [15]. To and IFNγ (Table 1). At Day 2 after LPS treatment, most of determine the physiological function and tumorigenesis these cytokines returned to basal levels in WT mice, but of XB130 in vivo, XB130 knockout (KO) mice have the levels of MCP-1, TNF-α, IL-6 and IL-10 in Xb130 KO been generated. XB130 deficiency could slow down mice were significantly higher compared to those in WT the differentiation of proliferating basal cells to ciliated mice (Figure 2A and 2B). epithelial cells during the repair process in trachea [16]. XB130 also promotes bronchioalveolar stem cell and Club XB130 deficiency led to more severe lung injury cell proliferation in small airway repair and regeneration [17]. The objective of the present study is to determine the Acute lung injury is one of the severe complications role of XB130 in septic response and acute lung injury. of sepsis [19]. To determine the role of XB130 in LPS- Acute lung injury (ALI) and its severe form, acute induced lung injury, a separate group of animals was given respiratory distress syndrome (ARDS), are critical and LPS, or PBS as a negative control. Again, at Day 2 after leading cause of mortality in the Intensive Care Units. LPS treatment, Xb130 KO mice revealed a significant In United States, there were approximately 190,000 weight loss (Figure 3A). Significantly higher composite patients of ALI/ARDS each year with approximately disease scores were found both at Day 1 and Day 2 after 40% mortality [18]. ALI/ARDS can be caused by LPS challenge in KO mice (Figure 3B). H & E staining of intrapulmonary insults (such as infection, acid aspiration, lung tissue sections revealed more severe lung injury in ventilator-induced lung injury, ischemia-reperfusion, Xb130 KO mice at Day 2 (Figure 3C), with significantly and etc.), as well as by extrapulmonary insults (such as higher lung injury score (Figure 3D) and wet/dry lung sepsis, hemorrhagic shock, trauma, burn, and etc.) [19]. weight ratio (Figure 3E), indicating severe lung injury and Lipopolysaccharide (LPS) binds to Toll-like receptor 4, pulmonary edema. and induces the production of various pro-inflammatory The spleens harvested after 6 hours showed normal cytokines and development of ALI [20]. pulp and peri-arteriolar lymphoid cells in mice that Multiple intracellular signal transduction pathways received PBS. The spleens from KO and WT mice that mediates ALI/ARDS [21]. For example, PI3K/Akt, received LPS were congested characterized by expanded mitogen-activated protein kinase, c-Jun N-terminal red pulp relative to the peri-arteriolar lymphoid cells. After protein kinase and p38 activation are involved in the 48 hours the spleens of LPS-treated mice in both the KO overproduction of reactive oxygen species, inflammatory and WT group showed less congestion and appear similar response, cell death and alveolar epithelial cell injury [22]. to the 6 hour PBS spleens. At Day 2, the glomeruli and Since XB130 is an upstream signal of Src, and PI3K/Akt, tubules of the renal cortices show preserved architecture. we used Xb130 KO and wild type (WT) mice to determine The liver parenchyma was also intact with normal portal the role of XB130 in LPS-induced septic response and tracts and lobular architectures. No significant differences ALI. were noted between WT and KO groups in LPS-treated animals in the spleen, kidney and liver (Supplementary RESULTS Figure S1), suggesting the differences observed in the lung are organ-specific. Xb130 KO mice were more susceptible to At Day 2 after LPS treatment, more numbers of LPS-induced septic responses apoptotic (TUNEL+) cells in the lung were observed in Xb130 KO mice compared to WT mice (Figure 4A To examine the role of XB130 in LPS-induced and 4C). By contrast, immunohistochemistry staining for systemic septic response, WT and Xb130 KO mice were Ki-67, a marker for proliferative cells, was significantly challenged with 25 mg/kg LPS. Seven days’ survival of less in Xb130 KO mice (Figure 4B and 4D). Xb130 KO mice (60%) was lower than WT mice (90%), At Day 2 after LPS treatment, the number of even though it did not reach statistical significance neutrophils (Ly-6B.2+) in the lung tissue was higher in (p = 0.11) (Figure 1A). Xb130 KO mice revealed a Xb130 KO mice (Figure 5A), but did not reach statistical significant weight loss at Day 2 (Figure 1B). Composite significance (Figure 5C). On the other hand, the number

www.impactjournals.com/oncotarget 25421 Oncotarget Figure 1: XB130 deficiency increased LPS-induced septic response in mice. (A) Kaplan-Meier survival curves of WT mice and Xb130 KO mice after LPS treatment (25 mg/kg). Difference in mortality was evaluated according to log-rank test (p = 0.114). Weight loss (B) and composite disease score (C) were recorded on different days. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout.

Figure 2: XB130 deficiency enhanced LPS-induced cytokine production. (A and B) Cytokine/chemokine levels in serum were measured at 2, 6 and 48 hours after LPS treatment. *p < 0.05 and **p < 0.01 (WT vs. KO), #p < 0.05 and ##p < 0.01 (PBS groups vs. LPS groups), WT = Wild type, KO = Xb130 knockout. www.impactjournals.com/oncotarget 25422 Oncotarget of macrophages (F4/80+) in the lung was significantly septic response, we challenged animals with LPS elevated in Xb130 KO mice compared to WT mice intraperitoneally. We observed significant differences (Figure 5B and 5D). between Xb130 KO and WT mice, in terms of body weight In Xb130 KO mice, more p-GSK-3β positive cells loss and composite disease score after LPS treatment. were observed in the alveolar spaces (Figure 6A and Moreover, the serum levels of several cytokines at Day 2 6B). To determine in which cell types p-GSK-3β positive of LPS treatment were significantly higher in Xb130 KO staining was evident, co-IF staining for p-GSK-3β together mice. MCP-1 is a CC chemokine for the recruitment of with markers for either macrophages (F4/8), neutrophils monocytes and other inflammatory cells to the sites of (Ly-6B.2) or type II pneumocytes (SFTPC) was tissue injury and infection [23, 24]. The pro-inflammatory performed. Figure 6C–6E revealed that both macrophages cytokines (TNF-α and IL-6) can activate neutrophils, and neutrophils, but not alveolar type II cells, were co- which adhere to the affected capillary endothelium and stained with p-GSK-3β. migrate into air spaces, leading to severe lung injury [25, 26]. IL-10 is an anti-inflammatory cytokine and plays Xb130 expression was reduced in the lung after protective roles in controlling immunopathology during LPS administration ALI [27, 28]. The expression of IL-10 can be rapidly induced by pro-inflammatory cytokines such as TNF-α XB130 is known to express in epithelial cells of [27, 28]. In this study, the levels of these cytokines in various human organs [3, 9, 16]. However, the expression serum increased at 2 and 6 hours after LPS treatment in of XB130 has not been assessed in mouse lung yet. To both groups, and then decreased in WT mice at Day 2, but detect the expression and location of XB130 in alveoli, remained significantly higher in the Xb130 KO mice. The co-IF staining for either Podoplanin (PDPN, a marker XB130 deficiency may not affect LPS challenge-induced for alveolar type I epithelial cell), or Surfactant protein rapid systemic inflammatory response, but may delay the C (SFTPC, a marker for alveolar type II epithelial cell), recovery. At Day 2, the histology of spleen, kidney and together with XB130 was performed. In WT mice, liver appeared relatively normal, which may be due to XB130 was expressed in the cytoplasm of both type I and the recovery of these critical organs from LPS challenge. type II alveolar epithelial cells (Figure 7). As expected, no The gene expression of XB130 is relatively higher in expression of XB130 was found in Xb130 KO mice (data the spleen [6], which is important for immunological not shown). XB130 staining was also observed in alveolar response. However, we did not see morphological epithelial cells in the human lung (data not shown). differences between KO and WT in the spleen, kidney and The Xb130 mRNA levels were significantly liver. Therefore, why serum cytokine levels in KO mice lower in the lung tissue of WT mice 2 days after LPS remaining high should be further studied. administration than that in the PBS control group Acute lung injury is one of the most common and (Figure 8A). XB130 was stained in alveolar epithelial severe complications in septic shock [19]. In the present cells in the PBS control group, but very low in LPS treated study, severe lung injury with more apoptotic cells, and group (Figure 8B). higher W/D weight ratio and less cell proliferation were observed in Xb130 KO mice at Day 2 after LPS treatment. DISCUSSION Cell death and inflammation may contribute to increased vascular permeability and tissue damage [29, 30]. Xb130, In this study, we showed that XB130 might play as an upstream regulator of PI3K/Akt pathway, specifically protective roles in LPS-induced systemic septic response binds p85α subunit of PI3K and leads to its activation and acute lung injury. [6]. Knocking down Xb130 with siRNA in thyroid Endotoxin-induced septic shock is the most or lung cancer cells reduced cell survival, increased common cause of morbidity and mortality in intensive susceptibilities of cells to extrinsic and intrinsic signal- care units. To determine if XB130 plays a role in systemic induced cell death, which was associated with increased www.impactjournals.com/oncotarget 25423 Oncotarget cleavage of caspase-8 and caspase-9. Down-regulation however, we did show expression of XB130 in the alveolar of XB130 also reduced cell proliferation and cell cycle epithelial cells. Thus, we would like to propose that the progression, together with reduced phosphorylation of lack of XB130 in lung epithelial cells may contribute to p21, p27, GSK-3β and FOXO3α [3, 7, 31]. XB130 is also apoptotic cell death in the lung after LPS stimulation, and required for cAMP-induced amplification of IGF related subsequently increase the pulmonary permeability and mitogenicity in thyroid cells [9]. We did not specify the edema. The reduced cell proliferation may explain the cell types that undergoing apoptosis or proliferation, delayed repair of lung epithelium.

Figure 3: XB130 deficiency enhanced LPS-induced lung injury. Weight loss (A) and composite disease score (B) were recorded for 2 days after LPS treatment. (C) Hematoxylin and Eosin staining showed severe lung injury in Xb130 KO mice at Day 2 after LPS challenge. Scale Bars = 50 μm. Lung injury score (D) and W/D weight ratio (E) were significantly higher in Xb130 KO mice. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout, W/D = wet/dry. www.impactjournals.com/oncotarget 25424 Oncotarget To determine whether the lack of XB130 expression protein level in cultured human bronchial epithelial cells affects PI3K/Akt pathway, we performed IHC staining (unpublished data); LPS may inhibit XB130 expression for phosphorylated p85α, Akt and GSK-3β on lung and reduce the protective effects of XB130 in ALI. This tissues. The staining of these proteins was very weak may also explain the weak staining of phosphorylation of on the alveolar wall in both WT and KO animals (data p85α and Akt and GSK-3β on alveolar wall. not shown). After LPS treatment, Xb130 mRNA level Interestingly, the numbers of p-GSK-3β positively was significantly reduced in the lung of WT mice, and stained macrophages and neutrophils were significantly the staining for XB130 was also extremely weak in higher in Xb130 KO mice after LPS treatment. GSK-3β is alveolar cells. LPS and TNF-α treatment reduced XB130 a serine threonine protein kinase, and a downstream target

Figure 4: XB130 deficiency resulted in more apoptosis and less cell proliferation in LPS-induced lung injury. (A) The number of apoptotic cells (red) was significantly higher (A–C), and the number of Ki-67+ cells (brown) was significantly lower (B and D) in Xb130 KO mice compared to WT mice at Day 2 after LPS administration. Scale Bars = 50 μm. *p < 0.05, **p < 0.01, WT = Wild type, KO = Xb130 knockout. www.impactjournals.com/oncotarget 25425 Oncotarget Figure 5: The number of neutrophils infiltrated into the alveoli at Day 2 after LPS treatment was not significantly different between WT mice and Xb130 KO mice (A–C). On the other hand, there was significantly higher number of macrophages infiltrated into the alveoli in Xb130 KO mice (B) (D). The neutrophils and macrophages were immunohistochemically stained with antibodies for Ly-6B2 and F4/80 antibodies, respectively. Scale Bars = 50 μm. *p < 0.05, WT = Wild type, KO = Xb130 knockout.

Figure 6: (A, B) Immunostaining for p-GSK-3β showed the number of p-GSK-3β+ cells (brown) were significantly higher at Day 2 after LPS treatment in Xb130 KO mice compared to WT mice. Scale Bars = 50 μm. p-GSK-3β was mainly stained in neutrophils and macrophages (C, D) but not in alveolar type II cells (E). *p < 0.05, WT = Wild type, KO = Xb130 knockout. www.impactjournals.com/oncotarget 25426 Oncotarget of PI3K/Akt pathway. Human monocytes stimulated with prolonged injury and delayed the repair of lung epithelial E. coli LPS demonstrated increased Ser 9 phosphorylation cells. The accumulation of macrophages in the lung tissue of GSK-3β [32]. The higher numbers of p-GSK-3β in Xb130 KO mice may contribute to the pathogenesis of positive macrophages and neutrophils may be due to ALI [33–35].

Figure 7: Immunofluorescence staining shows XB130 expression (XB130+: red) in alveolar type II cells (Sftpc+: green) (A) and type I cells (PDPN+: green) (B). PDPN = podoplanin, SFTPC = surfactant protein C. www.impactjournals.com/oncotarget 25427 Oncotarget In conclusion, our results indicate that the presence cell proliferation, and slows down the repair process. of XB130 is protective to systemic septic response and XB130 expression can be inhibited by LPS challenge. related acute lung injury. Its deficiency reduces cell Other ALI animal models should be used to further survival, promotes the infiltration of inflammatory cells explore the roles of XB130. Moreover, the role of XB130 into alveoli, increases pulmonary permeability, reduces should be addressed directly in human ALI.

Figure 8: (A) In WT mice, Xb130 mRNA levels were significantly reduced in the lung tissue 2 days after LPS treatment. (B) Immunofluorescence studies shows that the intensity of XB130 (red) expression in alveolar epithelial cells was extremely weak after LPS administration. WT = Wild type.

www.impactjournals.com/oncotarget 25428 Oncotarget MATERIALS AND METHODS Immunohistochemistry (IHC) and Immunofluorescence (IF) staining Animal care The primary antibodies used were: rabbit anti- The study protocol was approved by the Animal Use XB130 (1:1,000, Abgent, San Diego, CA), rabbit anti- and Care Committee of the University Health Network. Ki67 (1:100, Lab Vision, Fremont, CA), rat anti-Ly-6B.2 All animals were housed in a pathogen-free facility, (1:10,000, AbD Serotec, Raleigh, GC), rat anti-F4/80 with a 12-h light-dark cycle, and they were provided (1:500, AbD Serotec), goat anti-podoplanin (PDPN) with free access to water and food, and received humane (1:50, R & D Systems, Minneapolis, MN), rabbit anti- care in compliance with the Guide for the Care and Use surfactant protein C (SFTPC, for type II cells)(1:1,000, of Experimental Animals formulated by the Canadian Seven Hills Bioreagents, Cincinnati, OH) and rabbit Council on Animal Care. anti-phospho-GSK-3β-Ser9 (1:500, Cell Signaling, Xb130 KO mice were generated in collaboration Beverly, MA). After incubation with primary antibodies, with Dr. Tak W. Mak (University of Toronto) and sections were incubated with appropriate secondary maintained on a C57BL/6 genetic background. Xb130 KO antibodies. IHC was performed using a Vectastain ABC mice showed a normal life span, and did not have obvious kit (Vector Laboratories, Burlington, Canada) with phenotypes in a series of physiological testing compared 3-3-diaminobenzidine as chromogen, and sections were to wild-type (WT) littermates [16]. counterstained with hematoxylin, and images were captured via Olympus BX51-FL. For IF, the secondary LPS treatment and survival analysis antibodies used were: donkey anti-goat Alexa Fluor® 488, donkey anti-rabbit Alexa Fluor® 555 and goat anti- Sepsis and ALI were induced in female Xb130 KO mouse Alexa Fluor® 555 (1:200, Invitrogen, Burlington, mice and their female WT littermates (20 weeks of age) Canada), and sections were mounted with Prolong Gold by intraperitoneal injection of LPS (25 mg/kg, E. coli Antifade Mountant with DAPI® (Invitrogen). The slides serotype 055:B5, Sigma-Aldrich, St. Louis, MO). Two were examined with an Olympus BX-51, and images were groups of animals were used with 10 mice per group: 1) captured via QImaging colour camera (Olympus Co, Ltd). WT-LPS, 2) Xb130 KO-LPS. They were monitored for We randomly chose 5 fields (×200) per slide for positive 7 days after LPS treatment for survival, body weight and cell counting. composite disease score, based on the degree of ruffled fur, hunched appearance and lethargy [36]. Scores for each TUNEL assay item were defined as 0: not present, 1: mild, 2: severe. They were added with a maximum score of 6. Cell Death was assessed by in situ terminal transferase dUTP nick end labelling (TUNEL) with In Situ Assessment of acute lung injury and injury in Cell Death Detection Kit, TMR red (Roche, Penzberg, other organs Germany). The slides were examined with an Axiovert 200M microscope (Zeiss, Oberkochen, Germany), and Four groups of animals were used with 5 mice per images were captured via CoolSnap HQ camera (Roper, group: 1) WT-PBS, 2) WT-LPS, 3) Xb130 KO-PBS, 4) Ottobrunn, Germany). TUNEL-positive cells were Xb130 KO-LPS. At Day 2 after administration, mice quantified from 10 randomly chosen fields per slide were sacrificed, and the lung, spleen, kidney and liver (× 400) using ImageJ (1.46r). were harvested. The left lung and other tissues was fixed with 4% paraformaldehyde, and the upper lobe of the Quantitative RT-PCR for XB130 right lung was used for lung wet/dry (W/D) weight study, whereas the remaining lung tissue was snap-frozen and Total RNA was extracted using RNeasy kit stored at –80°C. The lung wet/dry (W/D) weight ratio (Qiagen, Duesseldorf, Germany). cDNA was synthesized was calculated by dividing the lung tissue weight before from total RNA using MuLV reverse Transcriptase and after drying it at 80°C for 48 hours. The lung and (Invitrogen). Quantitative RT-PCR was performed using other tissues were embedded in paraffin and cut at 5 μm SYBR Green I master PCR kit on Light Cycler480 thickness, and the sections were stained with hematoxylin (Roche Diagnostics). The Xb130 primer pairs used and eosin (H & E). Images were captured using Olympus were 5′- TCAGCATCTCCAGAC-3′ (forward) and BX51-FL (Olympus Co, Ltd). We randomly chose 5 5′-GGCTGTTTCCTCTCT-3′ (reverse). Each assay fields (×200) and assessed the lung injury with a modified included a standard curve of six serial dilutions and no- score system [37]. Briefly, the alveolar edema/exudates, template negative control. All assays were performed in hemorrhage, and interstitial/alveolar cellular infiltration triplicate. The relative expression level of Xb130 was were scored on a scale of 1–3 (0: absent, 1: mild, 2: calculated after normalization with housekeeping gene moderate, 3: severe) with a maximum score of 9 [38–40]. GAPDH. www.impactjournals.com/oncotarget 25429 Oncotarget Cytokine/chemokine measurement 6. Lodyga M, De Falco V, Bai XH, Kapus A, Melillo RM, Santoro M, Liu M. XB130, a tissue-specific adaptor protein For measurement of cytokine/chemokine, a separate that couples the RET/PTC oncogenic kinase to PI 3-kinase set of animals was used with 5 mice per group. The serum pathway. Oncogene. 2009; 28:937–949. was collected at 2, 6 and 48 hours after treatment, and 7. Shiozaki A, Shen-Tu G, Bai X, Iitaka D, De Falco V, bronchoalveolar lavage (BAL) fluid from the whole lung Santoro M, Keshavjee S, Liu M. XB130 mediates cancer was collected at 48 hours after treatment as previously cell proliferation and survival through multiple signaling described [41]. Cytokines/chemokines levels in serum events downstream of Akt. PLoS One. 2012; 7:e43646. and cell-free BAL samples were measured using Milliplex 8. Wu Q, Nadesalingam J, Moodley S, Bai X, Liu M. MAP Mouse Cytokine/Chemokine Magnet Bead Panel XB130 translocation to microfilamentous structures (MCYTOMAG-70K-12, EMD Millipore, Billerica, MA). mediates NNK-induced migration of human bronchial epithelial cells. Oncotarget. 2015; 6:18050–18065. doi: Statistical analysis 10.18632/oncotarget.3777. All values were expressed as mean ± standard error 9. Yamanaka D, Akama T, Fukushima T, Nedachi T, Kawasaki C, of the mean (SEM). 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